U.S. patent number 6,924,137 [Application Number 10/483,707] was granted by the patent office on 2005-08-02 for method for virus propagation.
This patent grant is currently assigned to Bavarian Nordic A/S. Invention is credited to Karl Heller, Paul Howley, Ingmar Rathe.
United States Patent |
6,924,137 |
Howley , et al. |
August 2, 2005 |
Method for virus propagation
Abstract
The present invention relates to a process for producing
poxvirus, in particular Chordopoxvirus, wherein the poxvirus is
cultivated at a temperature below 37.degree. C. The process leads
to increased virus propagation at the decreased temperature.
Inventors: |
Howley; Paul (Martinsried,
DE), Heller; Karl (Unterfohring, DE),
Rathe; Ingmar (Munchen, DE) |
Assignee: |
Bavarian Nordic A/S (Copenhagen
S, DK)
|
Family
ID: |
8160630 |
Appl.
No.: |
10/483,707 |
Filed: |
February 17, 2004 |
PCT
Filed: |
July 02, 2002 |
PCT No.: |
PCT/EP02/07280 |
371(c)(1),(2),(4) Date: |
February 17, 2004 |
PCT
Pub. No.: |
WO03/00853 |
PCT
Pub. Date: |
January 30, 2003 |
Foreign Application Priority Data
|
|
|
|
|
Jul 18, 2001 [DK] |
|
|
2001 01122 |
|
Current U.S.
Class: |
435/235.1;
435/239 |
Current CPC
Class: |
A61P
39/00 (20180101); A61P 31/12 (20180101); C12N
7/00 (20130101); C12N 2710/24051 (20130101); A61K
39/00 (20130101) |
Current International
Class: |
A61K
48/00 (20060101); A61P 31/12 (20060101); A61P
31/00 (20060101); A01N 63/00 (20060101); A61P
39/00 (20060101); C07H 21/00 (20060101); A61K
35/76 (20060101); A61K 35/66 (20060101); A61K
39/285 (20060101); C12Q 1/70 (20060101); A61K
39/12 (20060101); A61K 39/275 (20060101); C12M
3/00 (20060101); C12N 7/00 (20060101); C12N
7/01 (20060101); C12N 7/02 (20060101); C07H
21/04 (20060101); C12N 007/01 (); C12N
007/02 () |
Field of
Search: |
;435/235.1,239,320.1
;424/199.1,93.2 |
References Cited
[Referenced By]
U.S. Patent Documents
Other References
Drillien et al (Virology 119:372-381, 1982). .
Perkus et al. (Journal of Leukocyte Biology 58:1-13, 1995). .
Slonim D et al "Reaction of Infectious and lethal activities of
three strains of vaccinia virus to the Incubation temperature";
Journal of Hygiene, Epidemiology, Microbiology and Immunology; vol.
10, pp. 480 to 485 (1972). .
Slonim D et al "Influence of the Incubation temperature on the
dynamics of reproduction and plaque formation of three strains of
vaccinia virus in cell cultures"; Journal of Hygiene, Epidemiology,
Microbiology and Immunology; vol. 16, pp 474 to 479 (1972). .
Slonim D et al "Reproduction in chick chorioallantoic membrane and
lethal effect in chick embryos of three strains of vaccinia virus
in relation to the Incubation temperature"; Journal of Hygiene,
Epidemiology, Microbiology and Immunology; vol. 17, pp 21 through
25 (1973). .
Fuchs N et al "Virus Isolation and titration at 33.degree. C and
37.degree. C"; Med. Microbiol. Immunol.; 161, pp 123 to 126 (1975).
.
Mingle, Julius A.A. et al "Maintenance of primary african green
monkey kidney (pAGMK) and vero cells at room temperature
(25.degree. C). A system for virus isolation in community
practice"; Can. J Microbiol.; vol. 20, pp 391 to 397 (1974). .
Sutter, Gerd et al "Nonreplicating vaccinia vector efficiently
expresses recombinant genes"; Proc. Nat. Acad. Sci. USA; vol. 89,
pp 10847 to 10851 (Nov. 1992). .
Sugimoto, Masanobu et al "Characteristics of an attenuated vaccinia
virus strain LC16mO, and its recombinant virus vaccines"; Vaccine
1994; vol. 12, No. 8, pp 675 to 681..
|
Primary Examiner: Mosher; Mary E.
Attorney, Agent or Firm: Dubno; Herbert Myers; Jonathan
Parent Case Text
CROSS REFERENCE TO RELATED APPLICATION
The present application is the U.S. National Phase of
PCT/EP02/07280 filed 2 Jul. 2002 which claims the benefit of the
filing date of 18 Jul. 2001 of Danish Patent Application PA 2001
01122.
Claims
What is claimed is:
1. A process of amplifying a Chordopoxvirus characterized in that
the virus is propagated in culture media at a cultivation
temperature below 37.degree. C., wherein the chordopoxvirus is
selected from Avipoxvirus and modified vaccinia virus Ankara
(MVA).
2. Process according to claim 1, wherein the poxvirus is propagated
in chicken embryo fibroblast cells.
3. Process according to claim 1, wherein the Chordopoxvirus is
propagated at a cultivation temperature of about 26.degree. C. to
about 36.degree. C.
4. Process of amplifying a Chordopoxvirus characterized in that the
virus is propagated in chicken embryo fibroblasts at a cultivation
temperature of 26.degree. C. to 32.degree. C.
5. Process according to claim 4, wherein the Chordopoxvirus is
selected from the group consisting of Avipoxvirus and
Orthopoxvirus.
6. Process according to claim 5, wherein the Orthopoxvirus is a
vaccinia virus.
7. Process according to claim 6, wherein the vaccinia virus is
selected from strain Elstree and modified vaccinia virus Ankara
(MVA).
8. Process according to claim 1, wherein the Chordopoxvirus is
propagated at a cultivation temperature of about 26.degree. C. to
35.degree. C.
9. Process according to claim 1, wherein the Chordopoxvirus is
propagated at a cultivation temperature of about 30.degree. C.
10. Process according to claim 1, wherein the virus propagation is
performed for at least 24 hours.
11. Process according to claim 1, wherein the virus cultivation is
performed for at least 2 to 3 days.
12. Process according to claim 1, wherein the Chordopoxvirus is a
virus useful as a vaccine.
13. Process according to claim 1, wherein the process is performed
in a stationary flask.
14. Process according to claim 1, wherein the pox virus is not a
temperature sensitive mutant virus.
15. Process according to claim 1, wherein the pox-virus is not a
temperature attenuated virus.
16. Process according to claim 1, wherein no lipids or surfactants
are added to the culture media.
17. Process according to claim 1 wherein the modified vaccinia
virus Ankara (MVA) is MVA-BN as deposited at ECACC under No.
V00083008 or a derivative virus which shows the same temperature
dependency as the deposited strain.
18. Process according to claim 1 wherein the Chordopoxvirus is
propagated in avian cell culture.
19. Process according to claim 7 wherein the modified vaccinia
virus Ankara (MVA) is MVA-BN as deposited at ECACC under No.
V00083008 or a derivative virus which shows the same temperature
dependency as the deposited strain.
20. Process according to claim 6 wherein the vaccinia virus is
strain Elstree.
21. Process according to claim 4, wherein the Chordopoxvirus is
propagated at a cultivation temperature of about 30.degree. C.
22. Process according to claim 4, wherein the virus propagation is
performed for at least 24 hours.
23. Process according to claim 4, wherein the virus cultivation is
performed for at least 2 to 3 days.
24. Process according to claim 4, wherein the Chordopoxvirus is a
virus useful as a vaccine.
25. Process according to claim 4, wherein the process is performed
in a stationary flask.
26. Process according to claim 4, wherein the pox virus temperature
sensitive mutant virus.
27. Process according to claim 4, wherein the pox-virus is not a
temperature attenuated virus.
28. Process according to claim 4, wherein no lipids or surfactants
are added to the culture media.
Description
The present invention relates to a process for producing poxvirus,
in particular Chordopoxvirus, wherein the poxvirus is cultivated at
a temperature below 37.degree. C. The process leads to increased
virus propagation at the decreased temperature.
BACKGROUND OF THE INVENTION
The poxviridae comprise a large family of complex DNA viruses that
replicate in the cytoplasm of vertebrate and invertebrate cells.
The family of poxviridae can be divided into the subfamily
chordopoxvirinae (vertebrate poxviruses) and entomopoxvirinae
(insect poxviruses) (Fields Virology/eds.: Fields, B. N., Knipe, D.
M., Howley, P. M.; 3.sup.rd ed/ISBN 0-7817-0253-4/ see in
particular chapter 83).
The chordopoxvirinae comprise numerous animal poxviruses
(classified in different genera), such as camelpox-viruses,
sheeppox-virus, goatpox-virus or Avipoxviruses, in particular
fowlpoxvirus and also poxvirusus that are of relevance for humans
such as the variola virus and the vaccinia virus.
Pox-viruses, in particular chordopoxvirinae, are important
pathogens in humans and animals. There is also a long history of
vaccination against pox-virus infections. Nearly two centuries ago,
humans were prophylactically inoculated with cowpox to immunise
them against smallpox. Later immunisation was performed with the
Vaccinia virus. However, smallpox vaccination with this Vaccinia
virus resulted occasionally in serious complications, such as
postvaccinal encephalitis, generalised Vaccinia or contact
infection. Then, a new vaccine that does not show these
complications, was developed by Anton Mayr. The pox vaccine
consists of the poxvirus Modified Vaccinia Virus Ankara (MVA) and
was used for vaccination against smallpox in about 150 000
vaccinations without causing any complications related to the
vaccination. Even children with immunologic deficiencies did not
show serious side effects. The MVA was obtained by mutation and
selection of the original vaccinia virus Ankara after 575 passages
in chicken embryo fibroblast cultures. The safety of this MVA is
reflected by biological, chemical and physical characteristics. MVA
has a reduced molecular weight, six deletions in the genome, and is
highly attenuated for mammalian cells, i.e., DNA and protein is
synthesised but virtually no viral particles are produced.
The vaccination against smallpox was highly successful. In 1979,
the World Health Organisation declared the eradication of smallpox.
Accordingly, the mass vaccination of children was discontinued and
only laboratory workers and members of the armed forces of some
countries are vaccinated.
With the eradication of smallpox, the predominant cause of pox
viral infection in humans was removed. However, some non-human
poxviruses have reduced host specificity, i.e., they cause
infections not only in their typical host (e.g. for cowpox the
cow), but also in other animals, (e.g. rats and cats). Humans can
be infected by this route as well. Since parts of the population
are no longer immune against smallpox, poxvirus infections of
animal species can be dangerous for them. Domestic animals are the
main source of infection for humans. Accordingly, the vaccination
of domestic animals against poxviruses is. of increasing
importance. In addition, poxviruses are important vectors for the
expression of foreign genes for example for use as a vaccine or for
gene therapy, i.e. to transfer nucleic acid sequences into a target
cell where they are expressed. Consequently, an efficient and cost
effective production method for poxviruses is required.
Poxviruses can be amplified in different cell types. For example,
chordopoxvirinae, in particular MVA are amplified in cell cultures
of primary or secondary chicken embryo fibroblasts (CEF). The cells
are obtained from embryos of chicken eggs that are incubated for 10
to 12 days. The cells of the embryos are then dissociated and
purified. These primary CEF cells can either be used directly or
after one further cell passage as secondary CEF cells.
Subsequently, the primary or secondary CEF cells are infected with
the MVA. For the amplification of MVA the infected cells are
incubated for 2-3 days at 37.degree. C. (see, e.g., Meyer, H. et
al. 1991; J. of General Virology 72, 1031-1038; Sutter et al. 1994,
Vaccine, Vol. 12, No. 11, 1032-1040). Although other
chordopoxviruses are amplified in different cell types, the same
temperature of 37.degree. C. is chosen in those cases. For example,
the Vaccinia virus obtainable from ATCC (No. VR1354), which is
cultivated in HeLa S3 cells (human cervix carcinoma cells) is also
incubated for 3 days at 37.degree. C. (Current protocols in
molecular biology 1998, Chapter 16, Unit 16.16, John Wiley &
Sons, Inc). Furthermore, the MVA adapted for growing in Vero cells
(monkey kidney cells) is also amplified at 37.degree. C.
(PCT/EP01/02703). Consequently, independent from the cells used for
amplification and independent form the species or strain of the
chordopoxvirus, amplification of the viruses is performed at
37.degree. C. This selected temperature corresponds well with the
general knowledge of the skilled practitioner: Pox-viruses nearly
exclusively amplified in the laboratories are obtained from
warm-blooded animals with a body temperature of approximately
37.degree. C. Since chordopoxviruses are adapted for growing in
said animals, they are adapted for growing at 37.degree. C., i.e.
they should amplify most efficiently at 37.degree. C.
Because of similar reasons Entomopoxviruses are cultivated at
temperatures lower than 37.degree. C.: The body temperature of
insects is significantly lower than 37.degree. C. and depends to a
larger extent on the temperature of the environment. Thus, in
contrast to Chordopoxviruses the Entomopoxviruses are adapted for
growing at lower temperatures. U.S. Pat. No. 5,721,352 and U.S.
Pat. No. 5,174,993 disclose an optimal temperature for growth of
the Entomopoxvirus species Amsacta moorei Entomopoxvirus (AmEPV) of
28.degree. C. in the laboratory. However, these patents do not
disclose the cultivation of Chordopoxviruses under these
temperature conditions.
Furthermore, production of vaccines against other viral infections
is in general performed at 37.degree. C. Only some measles vaccines
are produced at a lower temperature. In this case, a measles
vaccine, which was originally produced at 37.degree. C. and which
frequently caused severe side effects, was attenuated by continuous
passaging of the virus at 32.degree. C. After 85 passages of the
strain at 32.degree. C. the strain was attenuated, i.e. the
disease-causing capacity of the virus was considerably reduced
(Plotkin, Orenstein: Vaccines, 3.sup.rd edition, 230-232). In
conclusion, viruses of warm-blooded animals and particularly
Vaccinia viruses are expected to amplify most efficiently at
37.degree. C., since they are found in animals with said body
temperature and adaptation to a lower temperature is only achieved
after multiple passages at said lower temperature. Furthermore,
adaptation to a lower temperature is associated with attenuation
and therefore often with reduced reproduction capacity of the
virus.
U.S. Pat. No. 5,616,487 discloses a process for producing a
stabilized virus, in particular a stabilized retrovirus, by
culturing virus producing cells with a stabilizing agent at a
temperature below 37.degree. C. The stabilizing agents are lipids
or surfactants. The patent specifically discloses Pluronic F-68 and
Lipid Concentrate as stabilizing agents. Lipid Concentrate is said
to contain cholesterol, cod liver oil, Pluronic F-68,
d-alpha-tocopherol acetate and Tween 80. In an alternative
embodiment U.S. Pat. No. 5,616,487 discloses a process for
cultivating specific retrovirus producing cells at a temperature of
lower than 37.degree. C., wherein the produced retrovirus is
stabilized using a stabilizer as defined above.
OBJECT OF THE INVENTION
It was an object of the present invention to provide a method for
preparing pox-viruses, in particular Chordopoxviruses which leads
to a higher yield of virus particles per infected cell.
DETAILED DESCRIPTION OF THE INVENTION
Said object is achieved by a process for preparing pox-virus, in
particular a Chordopoxvirus, wherein the virus producing cells are
cultivated at a temperature below 37.degree. C.
It was surprisingly found that the method according to the
invention leads to much more efficient amplification of the virus
at the lower temperature (below 37.degree. C.) which in turn
results in a higher virus yield relative to the number of infected
cells. Consequently, fewer cells are required to produce the same
amount of virus. This is especially advantageous for modified
Vaccinia Virus Ankara (MVA), since the production of CEF cells
required for MVA amplification is laborious and expensive.
Furthermore, the reduction of the incubation temperature allows
saving energy during the amplification process of the poxvirus and
hence saves costs in the production of the viruses.
The term "poxvirus" as used in the present application refers
preferably to poxviruses of the subfamily Chordopoxvirinae
(vertebrate poxviruses) (Fields Virology/eds.: Fields, B. N.,
Knipe, D. M., Howley, P. M.; 3.sup.rd ed/ISBN 0-7817-0253-4/ see in
particular chapter 83). The terms "Chordopoxviruses",
"Chordopoxvirinae" and "vertebrate poxviruses" are used
interchangeably in the present application. Preferred
Chordopoxviruses are poxviruses of the genera Orthopoxvirus,
Parapoxvirus, Avipoxvirus, Capripoxvirus, Lepripoxvirus,
Suipoxvirus, Molluscipoxvirus and Yatapoxvirus. Most preferred are
poxviruses of the genera Orthopoxvirus and Avipoxvirus.
In a preferred embodiment the pox-virus being produced by the
method according to the present invention is a pox-virus, in
particular a chordopoxvirus, which is useful as a vaccine or which
can be used as a gene therapeutic vector in order to introduce
genes of interest into a host cell. Suitable virus strains are well
known to the skilled person. Suitable strains can be obtained e.g.
from the American Type Culture Collection (ATCC) or the European
Collection of Animal Cell Cultures (ECACC).
As mentioned above, particularly preferred pox-viruses for being
produced according to the present method are Avipoxviruses and
orthopoxviruses. Examples for orthopoxviruses are vaccinia viruses;
such a the Vacciniavirus strains Elstree, Western Reserve, Wyeth,
NYVAC, NYCBOH, Paris, Copenhagen, more preferably the various MVA
strains and most preferably MVA-BN, deposited at the ECACC under
V00083008 or derivatives thereof. MVA-BN and its derivatives have
been described in detail in the PCT application PCT/EP01/13628,
entitled "Modified Vaccinia Ankara Virus Variant".
A "derivative" of the deposited virus is a virus, which shows
essentially the same growth characteristics, in particular the same
temperature dependency as the deposited strain but might differ in
at least one part of its genome.
The process according to the present invention can be carried out
with wild-type viruses, attenuated viruses and recombinant viruses,
respectively.
An "attenuated virus" is a virus originating from a pathogenic
virus but that upon infection of the host organism leads to a lower
mortality and/or morbidity compared to the non-attenuated parent
virus. Examples of attenuated poxviruses are known to the person
skilled in the art. An example for an attenuated Vaccinia virus is
strain MVA, in particular the strain that has been deposited at
ECACC with the deposition number V00083008 (see above).
The term "recombinant virus" refers to any virus having inserted
into the viral genome a heterologous gene that is not naturally
part of the viral genome. A heterologous gene can be a therapeutic
gene, a gene coding for a peptide comprising at least one epitope
to induce an immune response, an antisense expression cassette or a
ribozyme gene. Methods to construct recombinant viruses are known
to a person skilled in the art. The most preferred poxvirus vector
is MVA, in particular MVA 575 and MVA-BN (see above).
In contrast to all previous teachings in the prior art the
inventors of the present invention found that out of six
temperatures between 26.degree. C. and 37.degree. C. the
pox-viruses, in particular Chordopoxviruses, amplified the least
efficient at an incubation (cultivation) temperature of 37.degree.
C. It was surprisingly found that a method for the amplification of
pox-virus, in particular Chordopoxvirus, leads to higher yields of
virus if the virus producing cells are cultivated at a temperature
below 37.degree. C., preferably between 36.5.degree. C. and
26.degree. C. or between about 26.degree. C. and about 36.degree.
C., more preferably between 28.degree. C. and 33.degree. C., even
more preferably between 28.degree. C. and 32.degree. C., most
preferably at 30.degree. C.
Another preferred temperature range is 30.degree. C. to
36.5.degree. C. Particularly good results have been obtained in the
subranges 30.degree. C. to 35.degree. C., 30.degree. C. to
33.degree. C. and 30.degree. C. to 32.degree. C. The most preferred
temperature is 30.degree. C.
The term "about" in connection with temperature values refers
preferably to the specifically mentioned temperatures and to
temperatures being up to 0.5.degree. C. higher or lower than the
specifically mentioned temperatures. By way of example a
temperature of "about 30.degree. C. is to be interpreted as a
temperature in the range of 29.5.degree. C. to 30.5.degree. C.
In more general terms, in the process according to the present
invention the respective poxvirus is produced by cultivation of an
infected cell at a temperature which is lower than the body
temperature of the animal, including an human, that is the natural
host of the respective poxvirus. As far as Vacciniavirus is
concerned buffaloes are regarded as natural host (Baxby, D.:
Jenner's smallpox vaccine: the riddle of vaccinia virus and its
origin. London: Heinemann Educational; 1981: 1-214).
The cultivation of virus producing cells is preferably performed
for at least 24 hours, more preferably for at least 2 days or for
at least 3 days. Normally, virus free cells are grown at 37.degree.
C. until a sufficient amount of cells is obtained. Then the cell
culture is inoculated with virus and the temperature is then
reduced to the above-indicated temperature. In an alternative
embodiment the cell culture is brought to the above temperature
before being inoculated with virus.
The media used for the cultivation of the cells before infection
and for the production of virus using the process according to the
present invention may be the same or different. All media are
conventional standard media known to the person skilled in the art.
If necessary it is possible to add further additives such as
antibiotics, additional amino acids and/or foetal calf serum.
According to a preferred embodiment the media used in the process
according to the present invention do not contain lipids or
surfactants to stabilize the viral lipid envelope. More preferably,
the media used in the process according to the present invention do
not contain any of the following stabilizing agents: Pluronic F-68,
the combination of Pluronic F-68 and Tween 80.TM. or Lipid
Concentrate (Gibco/BRL, Gaithersburg, Md., catalogue no: 21900-014)
that contains cholesterol, cod liver oil, Pluronic F-68,
d-alpha-tocopherol acetate and Tween 80.
For the Chordopoxviruses cells are known to the person skilled in
the art that can be used in the process according to the present
invention. The type and nature of the cells is not critical as long
as the cells can be infected with the respective virus and as long
as progeny virus is produced from the infected cells. Preferably
the multiplicity of infection should be lower than 1.
Particularly preferred cells are vertebrate cells, e.g. mammalian
or avian cells.
According to a preferred embodiment the vertebrate cells that can
be used in the method according to the present invention for
Vaccinia viruses, in particular for the Vaccinia virus strains
Elstree and MVA, are Chicken Embryo Fibroblasts (CEF). It was
particularly unexpected that the process according to the present
invention can be used for CEF cells since chicken have a normal
average body temperature of 41.degree. C. Thus, the temperatures
used according to the present invention of below 37.degree. C.,
preferably of between 36.5.degree. C. and 26.degree. C., more
preferably of between 28.degree. C. and 33.degree. C., even more
preferably of between 28.degree. C. and 32.degree. C., most
preferably of 30.degree. C. are so different from the normal body
temperature of the chicken that one would have assumed that these
cells can not be used for the propagation of vaccinia viruses at
these temperatures. The same considerations apply for temperatures
in the range of 30.degree. C. to 36.5.degree. C., 30.degree. C. to
33.degree. C., 30.degree. C. to 32.degree. C., and in particular
for the temperature of 30.degree. C.
Moreover, the process according to the present invention is
preferably performed in stationary flasks.
A further advantage is that the process leads to increased yields
with "normal" virus strains, which do not require a temperature
sensitive mutation or a long and complicated attenuation to the
reduced temperature. In other words it is not required to use
temperature attenuated or temperature sensitive mutants in order to
achieve higher yields in virus particles at the lower temperature
compared to 37.degree. C.
It is known for temperature attenuated virus or temperature
sensitive mutants that they are amplified better at the lower
temperature; however, such strains normally are less reproductive
than non-attenuated or non-temperature sensitive mutants.
Therefore, the big advantage of the method according to the
invention lies in the fact that it can be applied to any kind of
normal, highly reproductive virus strain.
The virus prepared according to the present invention is preferably
used as a vaccine or for preparing a composition used in a gene
therapy protocol. Such applications of poxvirus are well
established in the art.
SHORT DESCRIPTION OF THE FIGURES
FIG. 1
In FIG. 1 single values from experiment 1 are performed in
duplicate (represented as dots). Bars represent the mean values.
For single values compare table 1.
FIG. 2
In FIG. 2 single values from experiment 2 are performed in
duplicate (represented as dots). Bars represent the mean values.
For single values compare table 2.
FIG. 3
In FIG. 3 dots are single values from the experiments with the four
different temperatures (experiment 3) performed in duplicate. Bars
represent the mean values. For single values compare table 3.
FIG. 4
In FIG. 4 dots are single values from the experiments with the four
different temperatures (experiment 4) performed in duplicate. Bars
represent the mean values. For single values compare table 4.
FIG. 5
In FIG. 5 bars represent the mean values of the experiments
performed in triplicates at 30.degree. C. and 37.degree. C. For
single values compare table 5.
SUMMARY OF THE INVENTION
The present invention relates to the following embodiments:
a process of preparing a poxvirus, in particular a Chordopoxvirus,
characterized in that the virus is propagated at a cultivation
temperature below 37.degree. C.;
the above process characterized in that the virus is propagated at
a cultivation temperature of about 26.degree. C. to about
36.degree. C.;
the above process characterized in that the virus is propagated at
a cultivation temperature of about 28.degree. C. to about
33.degree. C.;
the above process characterized in that the virus is propagated at
a cultivation temperature of about 30.degree. C. to about
33.degree. C.
the above process characterized in that the virus is propagated at
a cultivation temperature of about 30.degree. C.;
the above process characterized in that virus propagation is
performed for at least 24 hours;
the above process characterized in that virus cultivation is
performed for at least 2 to 3 days;
the above process characterized in that the pox-virus is a virus
useful as a vaccine or gene therapeutic vector;
the above process characterized in that the pox-virus is selected
from the group comprising Avipoxvirus and orthopoxvirus;
the above process characterized in that the virus is a vaccinia
virus;
the above process characterized in that the virus is modified
vaccinia virus Ankara (MVA), preferably MVA-BN as deposited at
ECACC under No. V00083008 or a derivative thereof;
the above process characterized in that it is performed in a
stationary flask;
the above process characterized in that the pox-virus is not a
temperature sensitive mutant virus;
the above process characterized in that the pox-virus is not a
temperature attenuated virus;
the above process characterized in that the virus is propagated in
chicken embryo fibroblast cells;
a virus prepared according to any of the above processes;
a composition containing the virus as above;
a vaccine containing the virus as above;
a composition for use in gene therapy comprising a virus genome of
the virus prepared according to any of the above processes;
use of a virus prepared according to any of the above processes as
a vaccine;
use of a virus proposed according to any of the above processes in
gene therapy.
EXAMPLES
The further examples further illustrate the invention.
Example 1
Effect of the Temperature on the Multiplication of MVA
Cell Culture Conditions
Primary CEF-cells were seeded in stationary flasks with a seeding
cell density of 2.times.10.sup.7 CEF cells/185cm.sup.2. Cells were
seeded in VP-SFM +4 mM L-Glutamine and 1% Antibiotics/Antimycotics.
At day four after seeding a cell density of 5.times.10.sup.7
CEF-cells/185 cm.sup.2 was assumed. The cells were infected with
0.1 TCID.sub.50 /cell MVA-BN (deposited at ECACC under deposit no.
V 00083008) by using RPMI w/o FCS. The infection, followed by
incubation for 72 h, was performed at 30.degree. C. and 37.degree.
C. (in experiments 1 and 2 with two different CEF preparations), at
30, 33, 35 and 37.degree. C. (experiment 3) and 26, 28, 30 and
33.degree. C. (experiment 4). The experiments were performed in
duplicate for each temperature. Virus replication was stopped by
scraping the cells into the media and by freezing media and cells
together at -20.degree. C. This mixture was freeze/thawed another
two times to mechanically release the virus from the cells. For the
experiments with 4 different infection temperatures virus
replication was stopped by freezing stationary flasks at
-20.degree. C. This mixture was freeze/thawed another three times
to mechanically release the virus from the cells.
Virus titres from every stationary flask were determined by using
an immunohistochemical assay. Infected cells were stained with a
Vaccinia virus specific antibody. Secondly, an HRP-coupled antibody
directed against the Vaccinia virus antibody was added. After
addition of a substrate infected cells appear in blue or brown
colour. Evaluation of the assay was done by using the formula of
Spearman and Kaerber determining the TCID50/ml (tissue culture
infectious dose). Experiments were performed in duplicate. As an
acceptance criterion for titration results MVA-BN standard with
known titre was used as an internal control for each titration
experiment. Results from the experiments were only taken when the
values from MVA-BN standard did not differ more-than .+-.0.5 logs
from the overall average.
Results
To include possible growth variations of primary CEF cells,
experiments were performed with two different cell preparations
when comparing infection temperatures 30.degree. C. and 37.degree.
C. The results from the two independent infection experiments are
shown in Table 1-2 and FIG. 1-2.
The results of experiments 1 and 2 show a clear increase in the
virus yield at 30.degree. C. compared to 37.degree. C. In
experiment 1 an increase of 0.5 logs and in experiment 2 of about
0.7 logs was achieved.
TABLE 1 In Table 1 values from experiment 1 are shown. Viral titre
[TCID.sub.50 / Viral titre [TCID.sub.50 / ml] at 30.degree. C. ml]
at 37.degree. C. Flask a 7.50E+07 3.20E+07 Flask b 1.30E+08
3.20E+07 Mean value 1.00E+08 3.20E+07
TABLE 2 In Table 2 values from experiment 2 are shown. Viral titre
[TCID.sub.50 / Viral titre [TCID.sub.50 / ml] at 30.degree. C. ml]
at 37.degree. C. Flask a 1.30E+08 3.20E+07 Flask b 1.30E+08
2.40E+07 Mean value 1.30E+08 2.70E+07
The data from the first 2 experiments are very promising that an
increase in the yield of MVA can be achieved by decreasing the
incubation temperature after infection. Therefore, it was decided
to go ahead and try also other temperatures to find an optimal
infection temperature. The used temperatures were 30, 33, 35 and
37.degree. C. The results from this experiment are shown in Table 3
and FIG. 3.
Comparing the yields of 37.degree. C. and lower infection
temperatures (35, 33, 30.degree. C.) showed a clear increase at
lower temperatures. The best viral titre [TCID50/ml] was obtained
in this experiment with an infection temperature of 30.degree. C.,
which was 0.8 logs higher compared to 37.degree. C.
TABLE 3 In Table 3 viral titres [TCID.sub.50 /ml] obtained at 30,
33, 35 and 37.degree. C. are shown. Viral titre Viral titre Viral
titre Viral titre [TCID.sub.50 /ml] [TCID.sub.50 / [TCID.sub.50 /
[TCID.sub.50 /ml] at 30.degree. C. ml] at 33.degree. C. ml] at
35.degree. C. at 37.degree. C. Flask a 1.30E+08 5.60E+07 3.20E+07
3.20E+07 Flask b 1.30E+08 1.00E+08 7.50E+07 1.30E+07 Mean 1.30E+08
7.50E+07 4.90E+07 2.10E+07 value
In the next experiment in stationary flasks even lower temperatures
than 30.degree. C. were tested to see if 30.degree. C. is really
the optimal temperature for multiplying of MVA in CEF cells.
Therefore, 26, 28, 30 and 33.degree. C. were used. The results from
this experiment are shown in Table 4 and FIG. 4.
TABLE 4 In Table 4 viral titres [TCID.sub.50 /ml] obtained at 26,
28, 30 and 33.degree. C. are shown. Viral titre Viral titre Viral
titre Viral titre [TCID.sub.50 /ml] [TCID.sub.50 / [TCID.sub.50
/ml] [TCID.sub.50 /ml] at 26.degree. C. ml] at 28.degree. C. at
30.degree. C. at 33.degree. C. Flask a 3.20E+07 1.00E+08 4.20E+08
1.80E+08 Flask b 1.00E+08 1.80E+08 2.40E+08 1.00E+08 Mean value
5.60E+07 1.30E+08 3.20E+08 1.30E+08
The highest yield in this experiment was found at an incubation
temperature after infection of 30.degree. C. Comparing the data
from the experiment before (4 temperatures between 30 and
37.degree. C.) and this one, 30.degree. C. was identified as the
optimal temperature for the incubation after infection for the
multiplying of MVA on CEF cells in stationary flasks.
Analysing the viral titers of the single temperatures from these
two experiments, it became clear that incubation at 37.degree. C.
might even give the lowest virus yield out of the 6 used
temperatures. The observed order for the yields at the used
temperatures is the following:
Example 2
Effect of the Temperature on the Multiplication of Vacciniavirus
Strain Elstree
In another approach Vaccinia virus strain Elstree was tested in
addition to MVA-BN for temperature-dependance. MVA-BN and Elstree
were multiplied on CEF-cells. For the experiment primary CEF-cells
were seeded in stationary flasks with a seeding cell density of
2E+07 CEF cells/175 cm.sup.2. Cells were seeded in culture media +4
mM L-Glutamine and 1% Antibiotics/Antimycotics. At day four after
seeding a cell density of 5E+07 CEF-cells/175 cm.sup.2 was assumed.
The cells were infected with 0.1 TCID.sub.50/ cell MVA-BN by using
RPMI w/o FCS and Elstree, respectively. The infection, followed by
incubation for 72 h, was performed at 30.degree. C. and 37.degree.
C. The experiment was performed in triplicates for each
temperature. Virus replication was stopped by freezing stationary
flasks at -20.degree. C. This mixture was freeze/thawed another
three times to mechanically release the virus from the cells.
Virus titers from every stationary flask were determined by
titration according to SOP/MVA/04. As an acceptance criterion for
titration results MVA F6 Standard with known titer was used as an
internal control for each titration experiment. Results from the
experiments were only taken when the values from MVA F6 Standard
did not differ more than .+-.0.5 logs.
The results from the infection experiments are shown in table 5 and
in FIG. 5.
The data of the experiment with MVA-BN show a clear increase in the
virus yield at 30.degree. C. compared to 37.degree. C. (0.667
logs). In the experiments with Elstree an increase of 1.125 logs at
30.degree. C. compared to 37.degree. C. was found.
TABLE 5 In table 5 virus titers [log.sub.10 TCID.sub.50 /ml]
obtained at 30 and 37.degree. C. for MVA-BN and Vaccinia Virus
strain Elstree are shown. Elstree Elstree MVA MVA 30.degree. C.
37.degree. C. 30.degree. C. 37.degree. C. CEF cells CEF cells Flask
a 9.0 7.88 7.25 6.375 Flask b 8.0 8.0 8 6.5 Flask c 8.25 7.38 7.5
6.5 Mean value 8.42 7.75 7.583 6.458
* * * * *